The present invention relates to pharmaceutical compositions and the use thereof in the stimulation of neuronal survival, proliferation and neurite elongation.
Fetal cells undergo differentiation and maturation under the influence of many known factors. Fetal cells exposed to various growth factors will differentiate into neuronal cells. Known factors that influence neural growth include Nerve Growth Factor (NGF), Brain-derived growth factor (BDGF), acidic and basic fibroblast growth factor (xcex1-FGF and xcex2-FGF, respectively), and Growth Promoting Factors 2 and 4 (GPF2 and GPF4). Once fully differentiated, neuronal cell survival may be influenced by various conditions and compounds. For example, under adverse conditions such as hypoxia, stroke and the like, factors such as purines released from dead or dying cells, as well as Interleukin-1 (produced by activated microglia), may play a role in neuronal survival and maturation.
In order to study these factors and their role in neuronal development and survival, a number of cell culture systems have been developed. One clonal cell line, termed PC 12, is derived from a rat pheochromocytoma tumor. Pheochromocytoma is a tumor of the adrenal medulla, a neural tissue, that secretes large amounts of adrenaline and noradrenaline. The PC12 cell line is routinely employed in cell culture systems used for the study of neuronal properties.
During the past three decades the number of molecules exhibiting trophic actions in the brain has increased drastically. These molecules promote and/or control proliferation, differentiation, migration, and survival (sometimes even the death) of their target cells. Such factors include neurotrophins, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors, ciliary neurotrophic factor and related molecules, glial-derived growth factor and related molecules, transforming growth factor-beta and related molecules, neurotransmitters, and hormones. Such factors are characterized by their trophic actions, their expression patterns in the brain, and molecular aspects of their receptors and intracellular signaling pathways. (See, e.g., Weisenhom et al., Int Rev Cytol 1999;189:177-265)
Nerve Growth Factor (NGF) is probably the most extensively studied growth factor. It is known to be involved in many morphological changes such as neurite outgrowth. NGF also helps support the survival of neurons in culture and presumably in vivo also. PC12 cells undergo differentiation into sympathetic neurons when treated with NGF, with about 20 percent of PC12 cells extending neurites in the presence of NGF. NGF also helps support the survival of neurons in culture and presumably in vivo also. NGF is not a ubiquitous neurotrophic factor, however. Within the peripheral nervous system, NGF does not appear to be a survival factor for parasympathetic neurons or developing motor neurons.
Adult neurons normally do not replicate. When cells undergo apoptosis, adenosine nucleosides and nucleotides are released extracellularly with the concentration reaching as high as 1 mM. High adenosine levels effect the development and character of the surrounding cells. One such effect is the stimulation of neurite outgrowth by the surrounding tissue. The ability of purines to stimulate neurite outgrowth in cultured cells has been studied extensively. The results have been mixed, sometimes yielding a positive result, and sometimes yielding a negative result.
Gysbers et al. (Int. J. Dev. Neurosci. 14(1):19-34 (1996)) report that neurite outgrowth of PC12 cells is promoted by the addition of extracellular GTP. Furthermore, GTP together with NGF synergistically enhanced neurite outgrowth. In the same study, the adenine nucleotides ATP, ATPxcex3S, ADPxcex3S and ADP inhibited, rather than enhanced, NGF-induced neurite outgrowth. Thus, the resultant action of purines on neuronal cells is unclear.
Neary et al. (TINS 19(1):13-18 (1996)) report that the trophic effects of purines on neurons are often synergistic with those induced by polypeptide growth factors. A synergistic enhancement of bFGF-induced neurite elongation was obtained with 2-chloroadenosine in cultured myenteric neuronal cells. No data was reported on whether guanosine enhanced bFGF-induced neurite elongation.
Neary et al. (Neurosci. Letters 242:159-162 (1998)) report that adenosine and ATP have mitogenic activity when added to fetal astrocyte cultures. There was a stimulation of DNA synthesis, and an activation of a mitogen-activated protein kinase termed ERK. However, they did not report any morphological changes in the astrocytes from exposure to adenosine or ATP, nor was the action of guanosine or its related nucleosides and nucleotides reported.
Immunophilins, receptor proteins that bind certain immunosuppressive drugs, have been reported to enhance neurite outgrowth in both PC12 cells and sensory neuronal cultures of dorsal root ganglia when binding ligands that are either immunosuppressive or non-immunosuppressive.
Ribavirin is an antiviral agent that also possesses immunosuppressant activity. However, the neurotrophic activity of Ribavirin and analogs thereof has not been previously recognized.
Thus, in view of the above, it would be advantageous to enhance the ability of growth factors to stimulate neuronal replication, increase neuronal survival, and stimulate growth of neuronal processes by an agent that is amenable to pharmacological manipulation. Furthermore, a non-naturally occurring molecule would be advantageous in order to increase selectivity for neuronal cells and potentially avoid other activities endogenous molecules may possess and thereby decrease the potential for adverse side effects.
The present invention is directed to compositions comprising 1-(xcex2-D-ribofuiranosyl)-1H-1,2,4-triazole compounds and either endogenous or exogenous neurotrophic factors. Methods of using such compounds and compositions are also provided.
In one aspect, the invention provides methods of using invention compositions nd compounds to enhance neurite outgrowth, neuronal survival and neuronal proliferation in mammalian cells, in vitro and in vivo. In a preferred embodiment, a 1-(xcex2-D-ribofuranosyl)-1H-1,2,4-triazole compound is infused directly to a desired situs. In a more preferred embodiment the 1-(xcex2-D-ribofaranosyl)-1H-1,2,4-triazole compound is administered orally.
In another aspect, the invention provides methods of treating a neurological disease in a mammal subject. In a further aspect, the invention provides methods of treating neuronal trauma in a mammal subject.